Membrane phospholipids play a crucial role in the pathophysiology of myocardial ischemic reperfusion injury. During the tenure of this proposal, it has become apparent that reperfusion of ischemic myocardium potentiates intracellular signaling via the activation of phospholipase C and D leading to the translocation and activation of multiple kinases including protein kinase C and MAPKAP kinase 2. Activation of kinases results in the induction of the expression of a variety of genes which play a significant role in the survival of the postischemic myocytes. As a spin off of this research, a novel role of phospholipids has become evidence, i.e., reperfusion of ischemic myocardium induces apoptosis, and membrane phospholipids are likely to play a significant role in the mechanism of the apoptotic cell death. The current proposal is based on this novel finding and represents a logical step for the continuing investigation. This proposed research will explore the role of phospholipids in reperfusion apoptosis by addressing the following specific aims: (i) down-regulation of aminophospholipid translocase and upregulation of lipid scramblase leading to the translocation of phosphatidylserine from the inner leaflet to the outer leaflet; (ii) reperfusion-mediated increase in cytosolic Ca2+ and free radicals in apoptotic cell death; (iii) relative importance of sphingnomyelin-sphingnosine-ceramide signaling and diacylglvcererol-protein kinase C/cyclin-dependent kinases signaling; and (iv) examine the regulation of inducer (Bax,Abl, fas, c-fos, c-myc) and repressor (bcl-2) genes (for apoptosis) in reperfusion injury and develop approaches to modulate induction of these genes in an attempt to reduce ischemia reperfusion injury. Since bcl-2 has been demonstrated as anti-death gene for apoptosis, transgenic mice over-expressing bcl-2 gene will be used to examine the altered signal transduction mechanism which could regulate apoptosis and responsible for the reduction of ischemic reperfusion injury. The ultimate goal of this investigation is to develop modalities to block apoptosis during reperfusion of the ischemic heart, which will hopefully ameliorate reperfusion injury.